PROJECT SUMMARY Development of neural circuits depends on a combination of molecular and activity-dependent processes. The activity can be spontaneous?such as retinal waves that course across the mammalian perinatal retina?or sensory-evoked. Spontaneous waves of retinal activity are necessary for circuit refinement in the brain. Although much is known about the retinal circuitry responsible for retinal wave generation, it is unclear how retinal waves shape the development of functional cortical architecture and what role they play in the emergence of functional cortical properties that are already established at eye opening, such as retinotopy and orientation selectivity. It is also completely unknown which subtypes of neurons participate in waves in visual cortex and when they are recruited during development. Here we will determine the roles and dynamics of excitatory and inhibitory neuron activity in visual cortex during the period of retinal waves using simultaneous cellular-resolution two- photon calcium imaging of genetically defined neurons within a local circuit and simultaneous wide field single- photon imaging of neuronal activity across cortex to assess the function of retinal waves in cortical circuit formation in awake, head-fixed mice. This novel multiscale, dual-imaging approach will bridge the gap between microscopic and macroscopic imaging in order to better understand the relationship between local cellular activity and mesoscale, cortex-wide activity. To identify interneurons and measure their participation in retinal wave activity in cortex, we will express the fluorescent reporter tdTomato in inhibitory interneurons using the cre/lox system in transgenic mouse lines expressing the green calcium indicator GCaMP6s under the pan- neuronal snap25 promotor. We will also assess the ontogeny of the spatiotemporal activity pattern of Nkx2.1-, somatostatin- (SOM), and vasointestinal peptide-expressing (VIP) interneurons and excitatory/inhibitory coupling on the mesoscale level during stage II and stage III retinal wave activity in visual cortex using viral expression of jRCaMP1b in interneurons and GCaMP6s in excitatory neurons. We hypothesize that cortical interneurons shape the flow of retinal wave activity in visual cortex and contribute to the emergence of retinotopy and orientation selectivity. We will test this by chronically silencing these cells using GiDREADD, then assessing spatiotemporal dynamics of retinal wave generated activity in visual cortex, in addition to mapping retinotopy and measuring orientation tuning using visual stimulation at eye opening. Furthermore, we will determine the specific roles of Nkx2.1, SOM, VIP interneurons during stage II and stage III retinal wave activity in visual cortex. This work will develop and apply essential tools for detailed investigation of the impact of specific neuronal populations on the functional organization of brain circuits, and will describe how the developing cortex propagates spontaneous activity, and how the functional connectivity between different subtypes of cortical neurons is sculpted by early spontaneous activity. This is critical to understanding normal brain development and dysfunction associated with neurodevelopmental disorders.